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Injectable Thermo-Sensitive Polypeptide-based CDDPComplexed Hydrogel for Improving Localized Antitumor Efficacy Shuangjiang Yu, Dianliang Zhang, Chaoliang He, Wujin Sun, Rangjuan Cao, Shusen Cui, Mingxiao Deng, Zhen Gu, and Xuesi Chen Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01374 • Publication Date (Web): 15 Nov 2017 Downloaded from http://pubs.acs.org on November 21, 2017

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Injectable Thermo-Sensitive Polypeptide-based CDDP-Complexed Hydrogel for Improving Localized Antitumor Efficacy Shuangjiang Yua,b, Dianliang Zhanga,d, Chaoliang Hea*, Wujin Sunb, Rangjuan Caoc, Shusen Cuic, Mingxiao Dengd, Zhen Gub* and Xuesi Chena* a

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese

Academy of Sciences, Changchun 130022, China. b

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill, North

Carolina State University, Raleigh, NC 27695, USA. c

Department of Hand Surgery, China-Japan Union Hospital, Jilin University, Changchun, 130033,

China. d

Department of Chemistry, Northeast Normal University, Changchun, 130022, China.

Email: [email protected] (Chaoliang He), Tel: +86-431-85262116.

Email: [email protected] (Zhen Gu), Tel: (919) 515-7944.

Email: [email protected] (Xuesi Chen), Tel: +86-431-85262112.

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ACS Paragon Plus Environment

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Abstract In this study, a type of novel thermo-sensitive polypeptide-based hydrogel with tunable gelation behavior through changing the content of carboxyl groups was developed for the purpose of improving the cisplatin (CDDP) release behavior and enhancing the localized antitumor efficiency. The introduction of carboxyl groups in methoxy-poly(ethylene glycol)-b-(poly(γ-ethyl-L-glutamateco-L-glutamic acid) (mPEG-b-P(ELG-co-LG)) not only led to adjustable mechanical properties of the hydrogel, but also significantly reduced the burst release of the drug through the complexation between the carboxyl groups of polypeptide and CDDP. Furthermore, both the good biocompatibility and biodegradable properties of mPEG-b-P(ELG-co-LG) hydrogel were observed in vivo. Interestingly, the CDDP-complexed mPEG-b-P(ELG-co-LG) hydrogel exhibited significantly enhanced antitumor efficacy in vivo compared to the mPEG-b-PELG hydrogel loaded with CDDP without complexation, although lower cytotoxicity and IC50 of the CDDP-complexed hydrogel was observed in vitro. Overall, the new type of injectable CDDP-complexed hydrogel may serve as an efficient platform for sustained CDDP delivery in localized tumor therapy.

Keywords: Thermo-sensitive hydrogel, CDDP-Complex, Localized delivery, Sustained release, Cancer therapy

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1. Introduction

Side effects of the antitumor drugs are still one type of the key issues which markedly limited the positive outcome of clinical cancer treatment, even though the therapeutic efficacies in some types of cancers have been improved significantly and considerable positive research and clinical results have been reported recently1. Notably, the efficacy and safety of the drugs have been effectively improved, by changing the delivery strategy. For instance, through the development of various drug delivery systems (DDSs) for cancer therapy recently2-19. Hydrogel, a class of unique soft material with tunable physical properties, interconnected pore structure and the capability of protecting labile drugs, have been engineered in depth to act as localized drug depots. These depots could be implanted surgically or injected directly for controlled release of various therapeutic agents, such as small molecules, bioactive agents and live cells.20-26 Especially, thermo-responsive hydrogels, which could be implanted through a syringe at the targeted sites and form hydrogels in situ under the stimuli of body temperature with minimal trauma and pain, have showed exciting advantages as macroscopic drug delivery vehicles in recent years27-30.

Despite significant advances of these materials have been shown in most localized drug delivery systems, it’s still a challenge for sustained release of hydrophilic drugs due to the uncontrolled high initial burst release, which often causes localized tissue irritation, pain, toxicity, etc., once the local drug concentration exceeds the recommended safety dose 31. To improve this situation, considerable attempts have been made to adjust the drug release behaviors, such as by improving the mesh size and microstructure of the hydrogel, by introducing dynamical linkers between the drugs and

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materials through electrostatic interaction, ionic or covalent bonds, or by construction of hierarchical release systems.21, 32-34

Cisplatin (CDDP), a famous first-line chemotherapeutic drug, has been wildly used in clinical treatments for many types of cancers due to its broad antitumor capacity 35, 36. Nevertheless, obvious side effects has been the major drawback, which limits desired clinical outcomes37. To date, different types of DDSs have been exploited to overcome these unfavorable factors of CDDP 38-41. Especially, localized drug delivery systems based on hydrogel have been shown to be efficient in improving antitumor efficacy and reducing the systemic toxicity to normal organs through directly treating the lesion sites 22. But as a hydrophilic drug, the controlled and sustained release of CDDP in situ still remains a challenge due to its small size, relatively high water solubility and rapid diffusion behavior, which usually caused high initial burst release and unwanted localized side effects31. To overcome the above challenge, many strategies have been investigated to improve the localized antitumor efficacy of CDDP

42-45

. For example, Shen et al. prepared a thermo-gelling polymer-

platinum( Ⅵ ) conjugate based on methoxyl poly(ethylene glycol)-b-poly(D,L-lactide) polymer, rendering a long-term CDDP release behavior in vitro42. Casolaro et al. developed a Cisplatin/Hydrogel complex based on poly(N-acryloyl-L-phenylalanine) homopolymer, which showed a near zero-order release phase over seven days in vitro except for an initial burst effect at the first few hours43. Synthetic polypeptides are a class of attractive biomaterials that have been widely used in biomedical field owing to their excellent biocompatibility, biodegradability, unique structures and functionalities analogous to natural proteins

46-48

. Recently, different types of

polypeptide-based hydrogels have been developed for biomedical applications

49, 50

. In our previous 4


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work, a series of polypeptide-based hydrogel with good biocompatibility and degradability have been designed and prepared for drug delivery and 3D cell culture51-53. Nevertheless, up to now, CDDP-complexed hydrogel based on synthetic polypeptides has never been reported yet.

Herein, we developed a novel thermo-sensitive CDDP-complexed hydrogel based on a biodegradable and biocompatible polypeptide block copolymer, methoxy-poly(ethylene glycol)-b(poly(γ-ethyl-L-glutamate-co-L-glutamic acid) (mPEG-b-P(ELG-co-LG)), for localized tumor treatment (Scheme 1). And L-glutamic acid containing carboxyl pendant was introduced into the polypeptide block to regulate the gelation behavior of the copolymer and improve the cisplatin release behavior54. The gelation behavior, mechanical property of the hydrogel, and the drug release behavior in vitro were investigated, respectively. Furthermore, the biocompatibility and degradation behavior of these materials were studied to confirm the safety in vivo. Eventually, the antitumor efficiency of the CDDP-complexed hydrogel was investigated both in vitro and in vivo to verify the importance of controlling drug release in localized drug delivery systems.

2. Materials and methods

2.1 Materials

3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and poly(ethylene glycol) methyl ether (mPEG, Mn = 2000) were obtained from Sigma-Aldrich. L-glutamic acid, γ-Benzyl Lglutamate were purchased form Aladdin Industrial Co. Cisplatin (CDDP) was obtained from Shandong Boyuan Pharmaceutical Co., Ltd. The amino-terminated mPEG (mPEG-NH2), γ-Ethyl-Lglutamate N-carboxyanhydride (ELG-NCA) and γ-benzyl-L-glutamate N-carboxyanhydride (BLG5


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NCA) were obtained according to the previous method

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55 51

. The solvents purchased from Beijing

Chemical Industry Group Co. Ltd, such as tetrahydrofuran, N,N-dimethylformamide and toluene, were dried according to the previous work

56, 57

. Other reagents were purchased from Sinopharm

Chemical Co. Ltd., China, and used without further purification.

2.2 Synthesis and structural characterization of mPEG-b-P(ELG-co-LG)s

The mPEG-b-P(ELG-co-LG)s were synthesized according to the previous method except for the incorporation of BLG-NCA in the polypeptide blocks (Figure 1 A)

58

. Briefly, mPEG-NH2 was

dissolved in toluene, residual H2O was removed through azeotropic distillation for 5 hours. Then toluene was evaporated under reduced pressure. Anhydrous DMF was added to re-dissolve mPEGNH2. γ-Ethyl-L-glutamate N-carboxyanhydride (ELG-NCA) and γ-Benzyl-L-glutamate Ncarboxyanhydride (BLG-NCA) were added into the above flask under N2 atmosphere. The reaction mixture was then stirred at 25 oC with a N2 protection for 72 h. The methoxy-poly(ethylene glycol)b-(poly(γ-ethyl-L-glutamate-co-γ-benzyl-L-glutamate) block copolymer (mPEG-b-P(ELG-co-BLG)) was obtained through precipitation twice in 10 fold diethyl ether (Figure S1 A). After being dried under vacuum, mPEG-b-P(ELG-co-BLG) was completely dissolved in dichloroaceticacid (DCA), 33% HBr solution in acetic acid was then added into the reaction flask. The reaction was kept at 30 o

C for 1 hour with stirring. Then, the above mixture was precipitated into cold diethyl ether. The

crude product of mPEG-b-(PELG-co-PLG) was dissolved in DMF and dialyzed against H2O for 48 h at room temperature. After adjusting the pH of the obtained copolymer solution to around 7.5 using 0.1 M NaHCO3, the final copolymer was obtained through lyophilization. And the disappearance of peak f in Figure S1 B suggested that the benzyl ester group has been removed. With changes in the 6


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ratio of mPEG-NH2/ELG-NCA/BLG-NCA, the polymeric structures were tuned as summarized in Table 1. 1

H NMR spectra of mPEG-b-P(ELG-co-LG)s were determined on a Bruker AV 400 NMR

spectrometer using CF3COOD as a solvent. FT-IR spectra of the P1-P3 were recorded by a Bio-Rad Win-IR instrument. The gel permeation chromatography (GPC) was used to determine the molecular weights (Mn) and polydispersity indexes (PDI) with DMF as the eluent at a speed of 1.0 mL·min-1 at 50 oC. Circular dichroism (CD) spectra of the materials were recorded on an Applied Photophysics Chirascan CD spectrometer with the polymer concentration of 0.3 mg·mL-1 in water at room temperature.

2.3 Sol-gel transition behavior

mPEG-b-P(ELG-co-LG)s were dissolved in water at different concentrations and were allowed to stir for 24 h in ice-water bath. Thereafter, 300 µL of the copolymer solution was added to each vial with the inner diameter of 8 mm. Then the phase diagram of these materials was investigated by a test tube inverting method through increasing the temperature by 2 oC per step every 10 min. The temperature was recorded as the sol-gel transition point if no fluidity was observed within 30 s after inverting the test bottle58. Each data point was obtained from triplicate tests.

2.4 In vitro hydrogel degradation behavior

The mPEG-b-P(ELG13-co-LG3) hydrogels (0.5 mL) with different concentrations (6.0 wt% and 8.0 wt%) were prepared according to the above method in the vials with the diameter of 16 mm.

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After that, 3 mL of phosphate buffer saline (PBS) containing 0.25 wt% NaN3 was added into each vial gently. Then, the vials were incubated in the shaker at 37 oC. The medium in the vials were completely replaced by fresh PBS at the predetermined time intervals and the mass of the remaining gel was measured each time.

2.5 In vivo hydrogel degradation and biocompatibility

The SD rats (~ 200 g) were obtained from Laboratory animal center of Jilin University, China. And all mouse studies were performed under the protocols approved by the School of Life Sciences Animal Care and Use Committee of Northeast Normal University. The solution of P2 (8.0 wt %, 400 µL) was injected into the right back of SD rats. And the rats were sacrificed after different time intervals. The status of remaining gel in each rat was recorded, and the surrounding tissues were collected and stained by H&E to evaluate the biocompatibility in vivo.

2.6 In vitro drug release

The carboxylic acid groups in mPEG-b-P(ELG-co-LG)s were neutralized by NaHCO3 solution (0.1 N) to generate the ionized carboxyl groups and facilitate the formation of the CDDP-complex. CDDP was added into 8.0 wt% of P2 solution at the final concentration of 1 mg·mL-1. The solution was stirred for 24 h in ice-water bath to allow the formation of CDDP-carboxyl complex. Then, 0.5 mL of the drug-complexed copolymer solution was added into each vial with an inner diameter of 16 mm, and the drug-loaded hydrogels were formed after placing the vials into a water bath at 37 oC for 10 min. After that, 3.0 mL of PBS or phosphate buffer without NaCl (PB, 10 mM) containing 0.25 wt% NaN3 was added into each vial carefully as the release medium. Three parallel samples were 8


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kept on an orbital shaker at 37oC. After different pre-designed time intervals, 1.0 mL of the release medium was removed from each vial and 1.0 mL of fresh release medium was then added to the vial. The collected release medium was kept in divided EP-tubes in dark. The CDDP concentrations of all the collected samples were determined by ICP-MS (X series II, Thermoscientific, USA). Furthermore, mPEG45-b-PELG14-based CDDP-loaded hydrogel was prepared in the same method as a control group.

2.7 In vitro inhibition efficacy against tumor cells

Three different tumor cell lines including C26, HeLa and MCF-7 were used to investigate the relative inhibition efficiencies against tumor cells of the CDDP-loaded hydrogels by MTT assay in vitro. Generally, the cells were seeded in 24-well plates at 20,000 cells/well with 0.5 mL standard Dulbecco’s modified Eagle’s medium (DMEM). 50 µL of CDDP-loaded hydrogels with different drug concentrations were formed on transwell® insert chamber at 37 oC overnight and the chamber was then added into 24-well plate containing fresh DMEM. The MTT assay was performed after incubation of the cells with drug-incorporated hydrogel for 72 h. Each data point was obtained from three separate tests.

Besides, the live/dead cell staining assay was performed to evaluate the relative cytotoxicities of the CDDP-loaded hydrogel in vitro. Briefly, 0.2 mL of PBS solution containing 2.0 × 10-3 mM calcein AM and 4.0 × 10-3 mM propidium iodine (PI) was added into each cell seeded well and incubated for additional 0.5 h. The live cells were stained green with calcein AM and dead cells were stained red with PI, respectively. 9


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2.8 In vivo anti-tumor evaluation

The 6 week-old female BALB/c mice (Laboratory animal center of Jilin University, China.) inoculated with a C26 tumor xenograft model were used to evaluate the antitumor efficiency of the CDDP-loaded hydrogel in vivo. All the handling procedures of the mice were following the protocols of the School of Life Sciences Animal Care and Use Committee of Northeast Normal University. Generally, when the tumor volume reached ~ 80 mm3, the mice were divided into six groups (n = 6) randomly and weighted, then each group was treated with a single peritumoral injection of different formulations using a syringe (1.0 mL). The tumor growth was monitored every two days, and the ଵ

volume was calculated using the formula ܸ = ଶ ܽ × ܾ ଶ , which a and b represent the length and width of the tumor, respectively. The weights of the mice in each group were also recorded as an indicator of systemic toxicity of the treatments.

2.9 Histology and TUNEL assay The mice were sacrificed at the 18th day after the localized treatments with different agents. Then, the tumor, heart, liver, spleen, lung and kidney of the sacrificed animals were collected and deposited into 4.0 % (w/v) of paraformaldehyde, respectively. The tissue sections were made into 6.0 µm thick slices and stained by hematoxylin/eosin (H&E) for histological analysis. Additionally, the tumor tissue sections were labeled by the terminal nucleotidyl transferase mediated nick end labeling (TUNEL) assay 57.

2.10 Statistical analysis

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Using the Paired Student’s t-test, the difference between treated groups was analyzed statistically. The significant would be labeled if p < 0.05. The data were expressed as mean ± standard deviation from at least three separate experiments.

3. Result and discussion

3.1 Polymer synthesis and characterization

For the purpose of controlled release of CDDP, the amphiphilic polypeptides containing different ratios of free carboxyl groups were synthesized through ring-opening-polymerization (ROP) of comonomers ELG-NCA and BLG-NCA. The reaction was followed by the deprotection of the BLG residues in the presence of HBr (Figure 1A). The structure of the materials was characterized through 1

H NMR and GPC. According to the 1H NMR spectra (Figure 1B) and Table 1, three mPEG-b-

P(ELG-co-LG)s have been obtained, the degrees of polymerization (DP) of the polypeptide block changed from 13 to 18. Furthermore, the contents of L-glutamic acid units and the Mn showed an ideal gradient change from P1 to P3. Additionally, GPC analysis of the products showed unimodal distribution, indicating the successful synthesis of the mPEG-polypeptide block copolymers.

To investigate the influence of the carboxyl groups on the secondary structure, the mPEG-b(PELG-co-PLG) copolymers were analyzed by FT-IR and CD spectra both in solid state and water solution. As shown in Figure 1C, with increased content of carboxyl groups the absorption peak at 1627 cm-1 assigned to the β-sheet conformation gradually reduced and the peak at 1657 cm-1 ascribed to the α-helix structure increased. Meanwhile, the CD spectra exhibited a decrease of β-sheet conformation and increment of α-helix as the content of carboxyl groups in the polypeptide block 11


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increased. The result was consistent with the FTIR data (Figure 1D). These results suggested that the increase of the hydrophilic L-glutamic acid residues in the polypeptide block promoted the formation of α-helix conformation in mPEG-b-P(ELG-co-LG)s 52, 59.

3.2 Hydrogel formation and properties

The thermo-responsive gelation behaviors of P1-P3 were investigated with test tube inverting method and rheological analysis. The samples were coded Gel-1, Gel-2 and Gel-3, respectively. As shown in Figure 2A, the percentage of the hydrophilic carboxyl group showed an obvious influence on the sol-gel phase transition, and the increase in the content of carboxyl groups led to marked decrease in the gelation ability of the copolymers. Furthermore, rheological testing suggested that the hydrogels showed thermo-responsive variation in mechanical properties. As temperature increased to 5 oC over body temperature, the storage moduli (G’) of 8.0 wt% P1 and P2 solutions in PBS displayed marked increments. In contrast, the 8.0 wt% P3 solution did not form stable hydrogel until the temperature was increased over 60 oC (Figure 2 A). Moreover, it was found that the increase in the L-glutamic acid content led to a reduction in G’ at the same polymer concentration (Figure 2B).

The morphologies of the hydrogel were characterized by SEM (Figure 2 C). At the polymer concentration of 8.0 wt%, the polymer solutions (Figure 2 C, a and b) formed stable hydrogels at 37 o

C, except for P3 (Figure 2 C, c) which transformed into stable gel at much higher temperatures

(Figure 2 A). The microstructures of the Gel-2 with or without CDDP both showed a porous structure as observed by SEM. The interconnected porous microstructure may be beneficial to the drug transportation and release from the hydrogel. 12


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Additionally, the degradation behavior and biocompatibility of Gel-2 were investigated both in vitro and in vivo. As shown in Figure 2 D, the degradation behavior of hydrogel exhibited concentration and time dependence in PBS. The hydrogel with the lower polymer concentration (6.0 wt%) showed relatively faster degradation rate compared to the 8.0 wt% hydrogel, which may be due to the decreased stability of the physically crosslinked network with the lower polymer concentration. With further research, it was found that the P2 solution at the concentration of 8.0 wt% formed hydrogel shortly after subcutaneous injection into rats, and the time-dependent degradation behavior was observed in vivo. The hydrogels exhibited faster degradation rate in vivo compared to in vitro studies and completely disappeared at ~ 4 weeks post-injection. This phenomenon may be attributed to the bio-stimuli factors which accelerates the breakage of peptide bonds in physiological microenvironment, such as proteolytic enzymes etc. 58

3.3 Drug release behavior and tumor cell inhibition in vitro

The drug release behaviors of CDDP loaded hydrogel were investigated under different conditions in vitro. The influence of carboxyl group-containing L-glutamic acid residues on the release of CDDP from gel-1 and gel-2 were investigated in PBS at 37 oC (pH 7.4). As shown in Figure 3A, a certain degree of burst drug release behavior could be observed both from the drug-loaded Gel-1 (Figure 3a) and Gel-2 (Figure 3b) in the first several hours because of the fast diffusion of free CDDP from both samples. However, gel-2 showed an obviously slower drug release rate compared to gel-1 during the following 7 days, which may be caused by the complexation between CDDP and the carboxyl groups of mPEG-b-P(ELG-co-LG)s. Moreover, the CDDP release behavior was also markedly influenced by the chloride (Cl-) ion concentration. As shown in Figure 3 A and C, only 13


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around 9.8% of CDDP in gel-2 was released in phosphate buffer (PB, 10 mM) without NaCl during the period of 7 days. This result was likely caused by the combination effects of reduced solubility of CDDP in the absence of NaCl and the complexation between CDDP and the carboxyl groups.60, 61

Furthermore, the cytotoxicities of the blank hydrogel and drug-loaded hydrogel were evaluated against C26, HeLa and MCF-7 cell lines, respectively. As show in Figure S2, these materials exhibited good biocompatibilities in vitro. Furtherly, the viabilities of the cells incubated with CDDP-loaded Gel-2 were higher compared to those treated with free CDDP and CDDP-loaded Gel1 because of the delayed drug release behavior (Figure 3 B, C and D). As listed in Table 2, the IC50 values of the CDDP-loaded Gel-2 double those of free CDDP. Moreover, the live/dead staining tests against HeLa cells indicated that both Gel-1 and Gel-2 have good biocompatibilities as shown in Figure 3 E (b and c). Moreover, higher cell viability was observed in the group treated with CDDPloaded Gel-2 (Figure 3, E, f) compared to either the group treated with free CDDP or the group treated with CDDP-loaded Gel-1, which was consistent with the results of MTT assay.

3.4 Anti-tumor efficacy in vivo

To further study the anti-cancer efficacy of mPEG-b-P(ELG-co-LG)s hydrogel-based drug delivery device, several samples were used to treat the C26 tumor-bearing BALB/c mice by a single peritumoral injection in situ. As shown in Figure 4 A, significantly higher inhibition efficiency against tumor growth was exhibited in the group treated with CDDP-loaded Gel-2 at a CDDP dose of 5.0 mg·kg-1 (Figure 4 A, f). Interestingly, the treatment with CDDP-loaded Gel-2 at a relatively lower drug dose (2.0 mg·kg-1) (Figure 4 A, d) showed an antitumor efficacy comparable to the 14


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treatment with free CDDP at the dose of 5.0 mg·kg-1 (Figure 4 A, e). Similar results were obtained by the measurements of the tumor weights in different groups (Figure 4 B and D). The results suggested that the CDDP-complexed hydrogel could significantly improve the antitumor efficacy in vivo, likely attributing to their capacity of retaining more sustained CDDP release behavior.

Furthermore, histological examination was used to further evaluate the therapeutic efficacy of the CDDP-loaded hydrogel. The images of H&E stained tumor tissues after treating with CDDP agents presented cellular apoptosis in different degrees compared to the negative control groups (Figure 4 E). Notably, the H&E stained image for the group treated with CDDP-loaded Gel-2 (Figure 4 E, e) showed the largest range of dead cells. Moreover, the images by TUNEL staining of the tumor tissue exhibited the highest cellular apoptosis ratio in the group treated with CDDP-loaded Gel-2 which was similar to the results by H&E staining (Figure 4 E). The results implied that the inhibition of tumor growth by the treatment with CDDP-loaded hydrogel was attributed to the tumor cell’s apoptosis.

Additionally, to investigate the systemic side effects of the treatments, the body weights of mice were recorded during the course of the animal tests. As shown in Figure 4 C, the animal body weights of all the groups treated by localized injection of different formulations showed no obvious difference. Moreover, the H&E staining images indicated that the organs obtained from the drugtreated groups showed no obvious pathological changes compared to those from the PBS-treated group (Figure 5). Overall, the above results suggested that the localized drug delivery system based on the CDDP-complexed polypeptide-based hydrogel led to enhanced antitumor efficacy with low systemic toxicity in vivo. 15


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4. Conclusions

In this work, we developed a novel thermo-sensitive CDDP-complexed hydrogel based on mPEG-b-(PELG-co-PLG) for improving the localized antitumor efficacy of Cisplatin. The content of L-glutamic acid units within the polypeptide block not only changed the secondary structure of the hydrogels but also obviously influenced their gelation property. The incorporation of carboxyl groups which can form complex with CDDP in H2O led to a sustained release profile. It’s interesting to note that, although the CDDP-complexed Gel-2 hydrogel showed lower IC50 in vitro compared to both free CDDP and carboxyl-absent Gel-1 loaded with CDDP, the CDDP-complexed Gel-2 hydrogel exhibited the highest antitumor efficacy in vivo following treating the C26 tumor-bearing mice with a single peri-tumoral injection. Attributed to the enhanced localized antitumor efficacy and low systemic toxicity in vivo, the novel injectable CDDP-complexed polypeptide hydrogel holds potential as a sustained drug delivery system for localized solid tumor therapy. Supporting information available: 1H NMR spectra of mPEG-b-P(ELG13-co-BLG3) and mPEG-bP(ELG13-co-LG3), supporting data for the biocompatibility evaluation of mPEG-b-P(ELG-co-LG)s in vitro.

Acknowledgments

The authors are grateful for the financial support from the National Natural Science Foundation of China (NSFC 51773199 to S.Y., 51622307 to C.H., 51390484 to X.C., 51520105004 to X.C.), the Youth Innovation Promotion Association CAS to C.H., the Sloan Research Fellowship to Z.G. and

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the Scientific Development Program of Jilin Province (20170520126JH to S.Y., 20150311069YY to S.C.).

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Scheme 1. Schematic illustration of the mPEG-b-P(ELG-co-LG)-based injectable thermo-sensitive CDDP-complex Hydrogel for localized tumor therapy (A). The structure of mPEG-b-P(ELG-co-LG) (B).

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Figure 1. The synthesis and characterization of mPEG-b-P(ELG-co-LG)s. Synthesis route of mPEG-b-P(ELG-co-LG)s (A), 1H NMR spectra (B), the FTIR spectra (C) and CD spectra in water of P1 to P3 (D).

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Figure 2. mPEG-b-P(ELG-co-LG)s hydrogel formation and properties. The sol-gel transition phase diagram of P1-P3 with different concentrations (n = 3) (A); Storage modulus (G’) of P1-P3 at the concentration of 8.0 wt% (B). The morphology characterization of the hydrogels, photographs of the 8.0 wt% polymer solution of P1 (a), P2 (b), P3 (c) in PBS and P2-CDDP (d) in water at 37 oC; SEM images of lyophilized Gel-2 (e) and CDDP-complexed Gel-2- (f) hydrogels (Scale bar = 20 µm) (C). The degradation behavior in vitro of Gel-2 at different concentration (n = 3) (D). The biostimuli degradation behavior of Gel-2 at 8.0 wt% (a, b, c) in vivo and tissue biocompatibility of the in site formed Gel-2 with H&E staining of the surrounding skin at different interval points (E).

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Figure 3. Drug release behavior and cellular experiment in vitro. The drug release behavior at pH 7.4 from CDDP loaded Gel-1 in PBS (a), Gel-2 in PBS (b) and Gel-2 in PB without NaCl (c) (A); Viabilities of C26 (B), HeLa (C) and MCF-7 (D) cells incubated with drug loaded Gel-1 (a), drug loaded Gel-2 (b) and free CDDP (c) at different drug concentrations for 72 h (n = 3). The viability fluorescence images of HeLa cells following the treatment with PBS (a), Gel-1 (b), Gel-2 (c), free CDDP with a final drug concentration of 10.0 mg·L-1 (d), CDDP loaded Gel-1 with a final drug concentration of 10.0 mg·L-1 (e) and CDDP loaded Gel-2 with a final drug concentration of 10.0 mg·L-1 (f) for 24 h (E). Green fluorescence stained by calcein AM represented the live cells, and red stained by propidium iodine (PI) represented the dead cells, respectively. Scale bars = 50 µm. (* P < 0.05, *** P < 0.001) 26


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Figure 4. Anti-tumor efficacy evaluation. Group treated by PBS (a), 8.0 wt% Gel-2 (b), 8.0 wt% Gel-1 with a CDDP dose of 2.0 mg·kg-1 (c), 8.0 wt% Gel-2 with a CDDP dose of 2.0 mg·kg-1 (d), Free CDDP with a dose of 5.0 mg·kg-1 (e) and 8.0 wt% Gel-2 with a CDDP dose of 5.0 mg·kg-1 (f) in vivo. The tumor areas (A), tumor weight (B), body weight (C), representative tumor images (D) of treated mice bearing C26 tumor (n = 6). Ex vivo H&E staining and TUNEL assay of C26 tumor tissues (E). Blue dye represented nuclei while red one indicated matrix and cytoplasm in the H&E staining. The bright red and blue stains indicated the apoptotic cells and nuclei which stained by TMR red-dUTP and DAPI, respectively. Scale bars = 50 µm. (* P < 0.05, *** P < 0.001)

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Figure 5. Ex vivo H&E staining of the major organs. Groups treated with PBS (a), 8.0 wt% Gel-2 (b), 8.0 wt% Gel-1 with a CDDP dose of 2.0 mg·kg-1 (c), 8.0 wt% Gel-2 with a CDDP dose of 2.0 mg·kg-1 (d), Free CDDP with a dose of 5.0 mg·kg-1 (e) and 8.0 wt% Gel-2 with a CDDP dose of 5.0 mg·kg-1 (f), respectively. Scale bar = 50 µm.

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Code

Copolymers

P1 P2 P3

mPEG-b-P(ELG13-co-LG0) mPEG-b-P(ELG13-co-LG3) mPEG-b-P(ELG13-co-LG5)

Feeding molar ratio mPEG/ELG-NCA/LG-NCA 1/15/0 1/15/3 1/15/6

DP of ELG/LG a

Mn b

PDI b

13/0 13/3 13/5

3,900 4,300 4,600

1.08 1.05 1.27

Table 1. Characterization of the copolymers applied in this study. a

Determined by 1H NMR.

b

Determined by GPC.

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Table 2. IC50 value calculation with different cell lines. Cell Line HeLa C26 MCF-7

Free CDDP 2.01 1.53 0.81

IC50 (mg/L) Gel-1 + CDDP 2.47 2.67 1.05

Gel-2 + CDDP 4.89 4.60 1.64


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Injectable Thermosensitive Polypeptide-Based ... - ACS Publications

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